Development of an expression system which produces a recombinant protein is important in providing a source of supply for protein which is used in research or therapy. As to the expression system, that based on prokaryotic cells such as Escherichia coli and that based on eukaryotic cells including yeast (genus Saccharomyces, genus Pichia, genus Kluyveromyces, etc.) and animal cells such as mammalian cells have been used. Among them, an expression system based on animal cells or, particularly, mammalian cells is preferred for the manufacture of protein for the therapy. That is because, since the posttranslational modification of protein which occurs in mammals such as human sometimes deeply contributes in bioactivity of the protein, and since posttranslational modification similar to that in the object to which protein is administered is possible in an expression system based on mammalian cells, efficiency of the protein for therapy is able to be enhanced by using the expression system based on mammalian cells.
As to a method for establishing the cells which produce a recombinant protein, it is general that a gene construct which expresses the gene of the aimed protein is transferred into host cells and, from the resulting transformed cells, the cells where the gene construct is stably transferred into the genome of the host cells are selected. At that time, a drug-resistant gene which acts as a drug selective marker has been previously inserted into the above gene construct so that said gene is expressed under a promoter which is same as or different from that of the gene of the aimed protein and then the cells survived by means of the drug selection are selected as the cells into which the gene of the aimed protein is stably transferred. Expression level of the aimed protein greatly varies depending upon the region of the host cell genome into which the gene coding for the aimed protein is transferred, but it is usually impossible to control the transfer region. Accordingly, even when gene transfer is carried out, most of the cells do not express the gene of the aimed protein or the expressed level is low. Therefore, in obtaining the transformed cells where the aimed protein gene is expressed in a high level, the operation of selection of one or two cell strain(s) from one thousand to several thousands of cell samples has been conducted repeatedly for several months to one year whereby quite a lot of labor and time have been needed for the selection of transformed cells which express the aimed protein gene in a high level.
Under such circumstances, a method of attenuating expression or function of a drug selective marker has been developed for an efficient selection of transformed cells which express the aimed protein gene in a high level. When expression or function of a drug selective marker is attenuated, the transformed cells where the drug selective marker gene is transferred into a low expression region in the host cell genome are unable to fully express the drug selective marker (drug-resistant gene) whereby they are dead and only the transformed cells where the drug selective marker gene is transferred into a high expression region survive by the drug selection. In the survived transformed cells, there is a high possibility that the aimed protein gene exists adjacent to the drug selective marker gene and, therefore, the aimed protein gene is also transferred into the high expression site in the host cell genome and there is a high possibility that the aimed protein gene is expressed in a high level. It is expected that, when that is utilized, transformed cells which express the aimed protein gene in a high level are able to be efficiently selected.
Examples of a method for attenuation of expression or function of the drug selective marker include a method where herpes simplex virus thymidine kinase (HSV-tk) promoter having a weak transcription activity is used for expression of the drug selective marker (Non-Patent Document 1) and a method where expression of the drug selective marker is suppressed using a variant type promoter where the transcription activity level is attenuated as compared with the wild type (Patent Documents 1, 2). However, when the present inventors carried out the follow-up studies of those methods, any of them rarely showed an effect in view of attenuating the expression of the drug selective marker. Therefore, it was thought to be difficult to efficiently select transformed cells which express the aimed protein gene in a high level using such a method.
Another method for the attenuation of expression or function of a drug selective marker is that mutation is transferred into the coding sequence of the drug selective marker such as neomycin phosphotransferase whereby the function of the drug selective marker itself is attenuated (Patent Documents 3, 4). The present inventors have carried out a follow-up study for this method and found that, when this method was applied, function of the drug selective marker was able to be attenuated to some extent and, as a result of the drug selection, small amount of cell strain exhibiting enhanced expression amount was able to be procured. However, the efficiency in terms of selection of transformed cells which express the aimed protein gene in a high level is not good and no significant effect was achieved by this method.
In establishing the highly productive cells, it is necessary to eliminate the influence from genome environment around the aimed gene-transferred site on the host cell genome for stably maintaining the expression of the aimed gene, in addition to the improvement in the cell selection art. Expression of the recombinant protein greatly varies depending upon the site of the host cell genome into which the gene coding for the recombinant protein is transferred. Usually however, control of the transfer site is impossible. Accordingly, even in the case of the cells exhibiting a sufficient expression upon screening, the expression sometimes lowers gradually as the cells are cultured continuously. In order to overcome such a position effect, a chromosome element (cis-acting DNA element) which mitigates the influence of the adjacent chromosome or regulatory element to the transgene has been utilized for the production of recombinant protein. One of the nucleic acid sequences having such a function is a sequence called an insulator and a DNAase I hypersensitive site of β-globin LCR of domestic fowls (cHS4; 1.2 kb length), etc. has been well analyzed for its function and utilized for the expression of recombinant protein (Non-Patent Document 2) but it has been reported that the increase in the protein expression in the CHO-K1 cells is not so significant (Non-Patent Document 3).